The study investigates the genetic differentiation among two subspecies of Allium ursinumL., namely A. ursinumsubsp. ursinumand subsp. ucrainicumas well as their putative hybrid that is represented by individuals withintermediate morphology. Inter-Simple Sequence Repeats (ISSR) were applied to determine the status of inter-mediate morphotypes in terms of their genetic pattern and to assess the level of genetic variability within andbetween various populations of A. ursinum. The study comprises 144 specimens from nine populations alongthe east-west transect in Poland, which includes localities of both subspecies and their putative hybrid. Amongthe examined populations, 48 bands were amplified, of which 45 were found to be polymorphic. The principalcoordinate analysis (PCoA), the neighbour-net analysis and Mantel test showed a strong correlation betweengenetic variability and geographic distance. Analysis of molecular variance (AMOVA) revealed that a greater pro-portion of total genetic variation resided within populations rather than among them. The Structure Bayesianclustering analysis revealed the presence of three distinct genetic groups within studied populations, where 'east-ern' genotypes correspond to A. ursinumsubsp. ucrainicum, and 'western' to subsp. ursinum; whereas the thirdgenetic group has the largest share in the individuals occurring at the border of the distribution ranges of bothsubspecies. The emergence of the third genetic group is probably an effect of hybridization events occurring with-in the secondary contact zone. Typical morphologically intermediate populations occur only in a relatively nar-row geographical zone, but the hybrid zone revealed by molecular markers is actually much wider than it is sug-gested by the morphological pattern of individuals. The current distribution pattern of both subspecies of A. ursinumand their hybrid zone is related to the two main directions of postglacial migration of Fagus syl-vaticato the area of Poland. The hybrid zone arose as an effect of the secondary contact of two divergent line-ages of A. ursinum.